The uses of oils are determined by their fatty acid composition. The principal component of oils are the triacylglycerol (TAG) molecules, which constitute normally more than 95% of the oil. Three fatty acids are bound to a molecule of glycerol to make the TAG. If these fatty acids are mainly saturated fatty acids (“saturates”) the product is called fat and it is solid at room temperature. On the other hand if the fatty acids are mainly unsaturated then it is called oil and it is liquid at room temperature.
The oils obtained from seeds cultivated in temperate climate (sunflower, soybean, rapeseed, etc.) have mainly unsaturated fatty acids, like linoleic and oleic acids, so they are liquid and primarily used for cooking, salad dressing, etc. Fats are obtained from animals (margarine, lard, etc.), some tropical trees (cocoa, palm) or chemically modified (hydrogenation and transesterification) liquid vegetable oils. They have mainly saturated (palmitic or stearic acids) or chemically modified fatty acids (trans fatty acids) all with high melting point.
Table 1 shows as an example the fatty acid composition and other properties of some fats and oils. The fats are needed for most of the food industry to make margarine, shortening, bakery, confectionery, snacks, etc. The food industry uses the fat for these purposes because of their plastic properties (they do not melt, can be spread, or do not stick to the hand) and stability (they have a good resistance to oxidation at room or high temperatures).
TABLE 1Fatty acid composition (%)PropertiesOil or fatOthers1MyristicPalmiticStearicOleicLinoleicTransSaturatedLard3225124510179Butter14102612283384Margarine107463423*Palm oil145539918Olive oil114371102Cocoa butter26353534Normal7530571sunflowerHigh548821oleicsunflower1“others” are palmitoleic in the case of lard and olive oil and also fatty acids shorter than 12 carbons in butter*depends on the level of hydrogenation
The actual available fats are however not a good option because they have negative nutritional properties. The main problem is that they raise the bad form of serum cholesterol (low density lipoprotein, LDL). This is due to several facts, some related to the origin of the fat and others with the manipulation thereof. Animal fats have most of the saturated fatty acids in the position 2 of the TAG molecule. Most vegetable fats and oils, however, have only minor amounts of saturated fatty acids in this position and are therefore more healthy.
During digestion the TAG molecule is hydrolysed by enzymes called lipases (FIG. 1). The fatty acids in positions 1 and 3 are liberated as free fatty acids. If these fatty acids are saturated they form insoluble salts with calcium and magnesium, being mostly excreted. But fatty acids in position 2 form with the glycerol a molecule of monoacylglycerol, which has detergent properties and is easily absorbed into the body. The saturated fatty acids from animal fats are then absorbed, thus raising LDL.
In order to increase the percentage of saturated fatty acids, vegetable oils are hydrogenated and/or transesterified. The hydrogenation process produces trans fatty acids that probably are even worse than saturated fatty acids as illustrated by Willett, W. C. & Ascherio, A. (1994) Trans fatty acids: Are the effects only marginal? American Journal of Public Health 84:722-724. The transesterification process changes randomly the fatty acids within the three positions, converting a healthy vegetable oil with low saturated fatty acid in the 2 position in an oil that has near 30% of saturated fatty acids. So neither of the two chemical modifications leads to a healthy product.
However, not all fats are unhealthy. It has been demonstrated that cocoa butter, which has around 60% of saturated fatty acids, the rest being mainly oleic acid, does not raise serum cholesterol. This is due to two main reasons. One is that only 4% of the saturated fatty acids are in position 2 and the other is that the principal saturated fatty acid is stearic acid. Stearic acid does not have a negative effect on serum cholesterol. Probably the amount of 35% of oleic acid in the cocoa butter also adds to its healthy property.
It is important to note that except in cocoa butter, palmitic acid is the main saturated fatty acid of commodity fats. Palmitic is however not a very healthy fat.
Traditional breeding and mutagenesis has not been the only tool used to form seeds producing oil with different fatty acid profiles. Increases in stearic acid in oil bearing plants have also been addressed by the introduction of transgenes into the germplasm, to alter the fatty acid biosynthesis pathway of the vegetable oil. The fatty acid biosynthesis in vegetable oil, but more particularly sunflower oil, includes the biosynthesis of basically two saturates (palmitate, stearate) and two unsaturates (oleate and linoleate). In oilseeds, the stearoyl-ACP desaturase is the enzyme which introduces the first double bond on stearoyl-ACP to form oleoyl-ACP. Thus, this is an enzyme that assists in the determination of the unsaturation in the C18 length fatty acids.
In U.S. Pat. No. 5,443,974 the inhibition of canola enzyme stearoyl-ACP desaturase was described. The stearate levels were increased but the levels of palmitate were basically unaffected. Inhibition of the plant enzyme stearoyl-ACP desaturase in canola was also reported by Knutzon et al., Proc. Natl. Acad. Sci. USA 89:2624-28 (1992). These results showed an increase in the level of stearate produced in the canola seed. The research also showed that inhibition by antisense in seeds of canola and soybean, respectively, showed increased stearate. When a plasmid containing a gene encoding for stearoyl-ACP desaturase was placed in canola, this inhibition resulted in an increase in stearic acid but unfortunately a reduction in the oleate. However, in the soybean this inhibition of stearate resulted in a less dramatic reduction of the oleate. This slower decrease in oleate however may have been a function of the small initial levels of oleate in the soybean. The fatty acid pathway in most oilseed plants appears to be resistant to maintaining both oleic and stearic at elevated levels.
PCT/US97/01419 describes increased levels of both stearic acid and palmitic acid in sunflowers through the inhibition of the plant enzyme stearoyl-ACP desaturase. As indicated above, palmitic oil is not, however, viewed as being a very healthy oil.
PCT/US96/09486 discloses that sunflower oil levels of both palmitic and oleic acids could be increased, the seeds having increased levels of palmitic acid of 21-23% and of oleic acid of 61%. The sunflower oil is liquid at room temperature. But the increased palmitic fatty acid level is alleged to allow the oil to be used in shortening and in margarine with relatively low level of hydrogenation, which leads to a relatively low level of trans-fatty acids in the resulting product. However, the commercial value may be questioned because of the high level of palmitic acid.
There thus remains a need for a sunflower oil which is both healthy and useful for industrial purposes. Furthermore, it is desirable to have a sunflower oil that has a balance of good saturates and good unsaturates, i.e. that is high in unsaturates but has sufficient saturates to be used for margarines or hardstock without high levels of hydrogenation, thus leading to no trans-fatty acids in the resulting product. Basically, there remains a need for a sunflower plant that can produce seed containing oil which is high in oleic acid and in stearic acid with reduced linoleic levels.